Perhaps the most rapidly developing area in separation science today is the burgeoning field of capillary electrophoresis (CE). The many unique features of this technology permeate all areas of chemistry, biology and medicine. Among the many unique features of capillary electrophoresis are its high power of resolution, high mass sensitivity, low sample volume requirements and overall sensitivity (Norberto A. Guzman Editor in Preface of Capillary Electrophoresis Technology. Marcel Dekker Inc. ISBN 0-8247-9042-1) .
High-performance capillary electrophoresis (HPCE) has achieved remarkably rapid development since its introduction in the middle 1980s. Today more than 10 companies offer automated instrumentation and sales are expected to grow to approximately 150 millions dollars in 1996.
CE can be used for the separation of biomolecules such as proteins, peptides and DNA. For example, polyanions such as heparin (Separation of natural and synthetic heparin fragments by HPCE. Damm et al., Journal of Chromatography 1992; 608: 297-309) and oligosaccharides derived from glycosaminoglycans (Honda et al., Journal of Chromatography 1992; 608:28) have been analyzed with CE. However, there are still many serious problems to solve, such as loss of efficiency, poor reproducibility of migration time and electroosmotic mobility because of sample-wall interactions (J. Kohr and H. Engelhaardt pp.357 in Guzman).
Indeed these biomolecules are polyelectrolytes, meaning that they contain both negative and positive charges, the net charge being positive (when there is an excess of positive charges) at a pH below their pI, neutral at a pH equal to their pI, or negative (where there is an excess of negative charges) at a pH above their pI. When analyzing a mixture of polyelectrolytes such as serum proteins at a mid-range pH, some proteins are positively charged and tend to stick to the wall of the capillary, causing bad reproducibility. To minimize this phenomenon when separating such a mixture, one works usually at an extreme pH. The pH can be, for example, below 2.5 so that all proteins are positively charged, with as a consequence the saturation of the capillary wall. This results in weak electroosmotic flow (EOF) but high mobility for the proteins. Alternatively, pH can be above 10 so that all proteins are negatively charged and are driven mainly by the high electroosmotic flow.
But electroosmosis has several disadvantages (Regnier and Wu Chap.8 in Guzman). First, it shortens capillaries by eluting analytes from the column before electrophoretic resolution is complete. This problem can be reduced by the use of longer capillaries but at the expense of the analysis time. A second disadvantage of EOF is that, when coupled with the adsorption of cationic proteins, it can cause band-spreading and poor reproducibility of migration time. In some situations adsorbed protein can even reverse the surface charge at the head of the column. The net effect is that the rate, and even the direction, of electroosmotic pumping at the capillary wall can vary along the length of the column. Subsequently, complex flow profiles develop within the column that compromise efficiency. In addition, axial differences in either ionic strength or pH which alter zeta potential can diminish efficiency and influence analyte transport velocity.
Electroosmosis is an electrically induced flow caused by the presence of an ion gradient at the silica/water interface which results in a slight excess of positive ions migrating towards the cathode. This then results in a net flow towards the cathode resulting flow rate gives an additional velocity component to the ions migrating in the electric field, such that the total velocity (v.sub.tot) of each molecule is the vector sum of the electrophoretic velocity (v) and the velocity imported by the electroosmotic flow v.sub.eo. V.sub.tot can be expressed as follows: ##EQU1## (t.sub.eo : observed migration time of electroosmotic marker); ##EQU2## (t.sub.tot : observed migration time of molecule). Mobility .mu. is equal to: ##EQU3## with E: electric field;
L.sub.d : capillary length from the anode to the detector; PA1 L.sub.t : total capillary length; and PA1 t: time of the analyte. PA1 .eta.: viscosity; and PA1 r: ionic radius. PA1 .DELTA. .mu..sub.el is the difference in electrophoretic mobility between two species to be separated; PA1 .mu..sub.eo is the electroosmotic mobility; and PA1 .mu..sub.el is the average electrophoretic mobility of the two species to be separated. PA1 1) by increasing the voltage, though this increase is limited by the instrumentation capacity and by the increase of amperage, generating Joule heating and subsequent higher convection; and PA1 2) by decreasing the diffusion coefficient by adding, for example, inert substances which result in longer migration times and also in higher Joule heating. PA1 rinsing a ready-to-use capillary with an initiator (dynamic rinsing), PA1 adding a capillary buffer into the initialized capillary, PA1 injecting a sample (possibly diluted with a sample diluent if needed) to be analyzed into the capillary, PA1 adding, optionally, a cathodic buffer to the cathode end of the capillary, PA1 submitting the sample to capillary electrophoresis, wherein the capillary and/or cathodic buffer comprise(s) a polyanion or a mixture of polyanions with the proviso that the polyanion or the mixture of polyanions is included at least in the capillary or in the cathodic buffer, and PA1 rinsing with NaOH after electrophoresis. PA1 B is H, OH, NH2, an alkyl, aryl, alkyl-aryl or heterocyclic skeleton and derivatives thereof, PA1 R.sup.1 to R.sup.m (i.e., R.sup.1 . . . R.sup.m) are monomers (m being a whole number greater than 1), R.sup.1 =R.sup.2 = . . . =R.sup.m or not, the resulting polymer being a homopolymer when R.sup.1 =R.sup.2 = . . . =R.sup.m or the resulting polymer being a heteropolymer wherein at least one R is different from the other R groups, R representing an alkyl, aryl, alkyl-aryl, or heterocyclic skeleton or group (with one or more nitrogen atoms) and their derivatives, or a nucleotide or nucleic acid group, an amino acid or peptide group, or phosphate group, PA1 A is H, OH, NH2, an alkyl, aryl, alkyl-aryl, a heterocyclic skeleton and derivatives thereof, PA1 B is H, OH, NH2, an alkyl, aryl, alkyl-aryl or a heterocyclic skeleton and derivatives thereof, PA1 W.sup.1 to W.sup.m are H, OH, O, an acidic radical, or an alkyl, aryl, alkyl-aryl, or heterocyclic skeleton bearing an acidic radical, PA1 Y.sup.1 to Y.sup.m are H, OH, an acidic radical or an alkyl, aryl, alkyl-aryl, heterocyclic skeleton bearing an acidic radical, an amine (primary, secondary or tertiary amine), a nitrogenous heterocycle or a mixture thereof, with the proviso that at the working pH the polyanion bears a net negative charge. PA1 A is H, OH, PA1 B is H, OH.
Consequently: EQU .mu..sub.tot =.mu.+.sub.eo
From this equation it can be seen that the electroosmotic flow adds the same velocity component to all analytes regardless of their ionic status. Consequently, a constant electroosmotic mobility (EOF) during analyses is necessary to obtain reliable analytical results from run to run, from day to day, from one capillary to another, or between different capillaries (when working with multiple capillaries).
The mobility .mu. is related to the charge and the ionic status of the analyte by the formula: ##EQU4## with q: net charge;
From this equation it can be seen that neutral substances have a mobility .mu.=0 and then move with a mobility equal to .mu..sub.eo. Therefore, neutral substances move with the same .mu..sub.tot =.mu..sub.eo whatever their radius or molecular weight and cannot be separated. From this equation it can also be seen that negative (or positive) substances of the same net charge and of the same radius but differing only by the position of a neutral functional group cannot be separated. In order to separate such neutral, negative, or positive substances, one must either make derivatives of such substances (pre-column derivatization or in-situ derivatization), these derivatives being charged, or one must use a buffer containing complexing or interacting species which can attract preferentially some functional group or polarizable group.
However, the velocity of the electroosmotic mobility is strongly dependent on many parameters, such as pH, ionic strength, the buffer composition and the chemical nature of the wall. The reproducibility of electroosmotic flow varies non-linearly with the pH, and the relative standard deviation (RSD) is usually higher between pH 4.5 and 7 than at lower or higher pH levels. (J. Kohr and H. Engelhardt J. Microcol. Sep. 1991;3:491)
The peak area in capillary electrophoresis is a function of the amount of sample present, its extinction coefficient, and the velocity of the solute peak. Unlike HPLC, the peaks in CE are travelling past the detector at different speeds. A slower-moving peak will have a greater-integrated area than a fast-moving peak, even if the extinction coefficient and amount of material are identical for each peak. Thus, a small change in the EOF may have dramatic effects on retention time (migration time) and peak area for both qualitative and quantitative applications (Tsai, Lee Direct control of EOF in CE by using an external electric field, p.476 in Guzman).
The total number of theoretical plates (N) is described by the equation ##EQU5## where V is the voltage and D is the diffusion coefficient.
Therefore the highest efficiency is obtained when a molecule is migrating at the fastest velocity (i.e., at the largest value of .mu..sub.tot). We can see that the electroosmotic flow helps speed up the separation in CE and thereby increases the separation efficiency. Further, the separation is directly proportional to the applied voltage (V) because a molecule that moves through the column quickly does not have much time to be spread out by longitudinal diffusion. Thus, working at high voltage and at the fastest velocity results in a very fast separation in the shortest analysis time. But such a short analysis time does not give the best separation. Indeed, the resolution (R) parameter that expresses the quality of the separation or the ability of the system to separate two closely eluting species is given by the following formula: ##EQU6## where: V, D are the applied voltage and the diffusion coefficient;
From this equation it can be seen that an increase can be obtained:
Once optimalized, all of these parameters are usually kept constant so that the only way to increase the resolution of closely migrating analytes is to decrease .mu..sub.eo in order to render the denominator of the third term in the formula above as low as possible. Higher resolution can be obtained when .mu..sub.eo =0, but the gain in resolution achieved by decreasing or canceling .mu..sub.eo will be obtained at a large expense in analytical time (Tsai, Lee in Guzman page 475). Furthermore, negative substances moving with a velocity v.sub.tot =v.sub.eo -v will migrate towards the anode once v becomes higher than v.sub.eo and then will not pass the detector.
Consequently, methods which use such a decrease or a cancellation of the electroosmotic mobility separate neutral and positive analytes. To resolve a mixture of neutral, negative and positive analytes, one must analyze such a mixture twice, either first with normal polarity and then with polarity reversal, or first at an acidic pH and then at a basic pH. Both methods generate two graphs, complicating or forbidding any quantification. Furthermore, when working in polarity reversal with v.sub.eo still positive, one cannot calculate the mobilities, since the neutral marker would not pass the detector. This leads to uncertainty in the identification of the peaks.
Various strategies have been devised to control or to suppress electroosmotic mobility using static coatings (permanent covalent chemical modification of the capillary surface by additives) or dynamic coatings by rinsing the capillary with additives before analysis or adding the additives in the buffer.
Kohr and Engelhardt (in Guzman, Chapter 10: CE with coated capillaries) conclude that capillary coating presents numerous problems such as reproducibility of coating procedures and the long-term stability of the capillary columns. No universal coating for the separation of proteins has yet been found. The following table is a compilation of their tables 1 and 2 with added data from the text:
__________________________________________________________________________ pH Functionality range Applications Effect on EOF Stability Page __________________________________________________________________________ Trimethylsilyl 7 small reduced hydrolysis at higher 363 molecules MECC pH Amylpentafluor 7 proteins retained SD 7.6% day to day 365 Polyethylene 3-5 proteins reduced no data at pH &gt; 7 glycol-Dial Polysaccharides 8 proteins reduced or NA 368 protein reversed Polyacrylamide 2-8 proteins suppressed not stable pH &gt; 8 370 Polyvinyl- 2-6 proteins suppressed 373 pyrolidone Polyethylen- 3-11 proteins reversed 375 cimine Poly (methyl- 1-9 proteins slightly 375 glutamate) reduced __________________________________________________________________________
Other additives used to decrease or to suppress the electroosmotic mobility, either dynamically or statically, are diamines and polyamines. These amines may be added to a buffer which does not contain any amine as the buffer itself and which is brought to the required pH by addition of a given acid. At a pH below their pI these amines are positively charged so that one positive end sticks to the negative wall of the capillary, exposing the other positive end(s) to the buffer. This suppresses the electroosmotic flow and can even reverse it.
Enantiomer separation is also an important field, not only in biochemistry but also in pharmaceutical analysis, where often one enantiomer is more active than the other and may be responsible for inadvertent side effects (e.g., Thalidomide-Softenon). Enantiomer separation by chiral capillary electrophoresis offers considerably greater efficiency with a shorter analysis time than GC or LC. Here again, screening of enantiomers by capillary electrophoresis requires more reliability than is presently available. Poor repeatability of the migration times due to the unsteady EOF is considered one of the most negative features of capillary electrophoresis. It is not likely that the development of coated capillaries and more standardized apparatuses will solve this problem (H. Siren, Jumppanen J, Manni Wen, Riekkola ML, Electrophoresis 1994; 15:779-84). These authors propose the use of two marker compounds to identify analytes on the basis of their mobility. However, chiral modifiers usually cause a decrease of the velocities and thus necessitate a longer analysis time.
In a recent paper (Dette C, Ebel S, Terabe S, Electrophoresis 1994; 15:799-803) a tetrakis (6-0-(4-sulfobutyl()-(-cyclodextrin sodium salt was used as a chiral selector. The authors note that these CDs, which are modified with negative groups, provide two features. They are still chiral selectors and have their own electrophoretic mobility opposite to the electroosmotic mobility. The latter could mean a better resolution when analytes with an opposite charge to the CD are used. The complex of the analyte with the negative CD derivative will have a negative mobility. For that reason a basic pH is necessary for good resolution.
Use of non-immobilized polyelectrolytes has also been described in capillary electrophoresis as a means to achieve chiral separation. For example, bovine serum albumin (Barker G, Russo P, Hartwick R, Anal Chem 1992; 64:3024-28), orosomucoid, ovomucoid, fungal cellulase and bovine serum albumin (Busch S, Kraak J, Poppe H, Journal of Chromatography 1993; 635:119-26) human albumin, bovine albumin (Vespalec R, Sustacek V, Bocek P, Journal of Chromatography 1993; 638:255-61), orosomucoid (Isaihama, Oda, Asakawa, Ijoshida, Sato, Journal of Chromatography 1994; A666:193-201) avidin in affinity electrokinetic chromatography (Tawaka, Matsubara, Terabe, Electrophoresis 1994; 15:848-53), and cellulase (Valcheva, Mohammad, Petterson, Hjerten, Journal of Chromatography 1993; 638:263-67) have all been proposed.
In a paper issued in October 1994 (Nishi, Nakamura, Nakai, Sato, Terabe, Enantiomer separation of drugs by affinity electrokinetic chromatography using dextran sulphate, Electrophoresis 1994; 15:1335-40), the authors used dextran sulphate (MW 7300) as a means to achieve chiral separations of some drugs at the expense of a decrease of the mobilities or an increase of the migration times not only of the solutes but also of the EOF marker as shown in their table 2.
______________________________________ Concentration of 0.5 1 2 3 4 5 dextran sulphate Mobility of methanol 3.617 3.438 3.392 3.375 3.351 3.142 ______________________________________
Furthermore, their FIG. 7 demonstrates that decreasing the pH of the buffer from 6 to 5.5 increases the migration times of the solute by about 60%.
In Micellar Electrokinetic Chromatography (MEKC), a micelle incorporating a neutral substance is negatively charged and migrates towards the anode, but if the electroosmotic mobility is higher than the mobility of the micelle, the latter will be driven towards the cathode. In ion-exchange electrokinetic chromatography, the separation principle is based on the differential ion-pair formation of the analyte with a polymer ion having a charge opposite (positive) to that of the analyte (negative). Accordingly, an analyte ion bonded to the polymer ion through ion-pair formation migrates in the opposite direction of the free analyte ion. It is consequently possible that even analytes having identical electrophoretic mobilities will migrate with different velocities if their ion-pair formation constants are different (Terabe J, Isemura T, Journal of Chromatography 1990; 515:667-76). The polymers used are polycations polybrene and poly (diallyldimethylammonium chloride), and they result in an electroosmotic mobility towards the anode. For some separations, such as negative analytes at low pH, a polarity reversal is required, meaning that the sample is injected at the cathode instead of the anode, the detector being at the anode instead of the cathode.
To enhance separation resolution and to prevent protein adsorption, Tsai and Lee (Direct control of EOF in CE by using an external electric field, P. 476 in Guzman) propose a physical method involving the use of an additional perpendicular electric field applied from outside of the capillary for the direct control of the EOF. According to its polarity, this external electric field can increase or decrease the EOF.
This instrumental approach present the following advantages: only a type of capillary, a direct and dynamic manipulation of EOF, a high degree of optimization for the separation efficiency and resolution is achieved, and a higher degree of automation is established. But this is achieved at the expense of a higher instrument cost, two perfectly isolated "power supplies" being needed both working in the range of 10000 volts. Such power supplies cannot be used with most existing instruments. Furthermore, this technique does not eliminate protein adsorption (Yoo, Wu and Regnier, Manipulation of EOF in CE, Journal of Chromatography 1993; 636:21-29).
The last and probably the most important problem of CE is the capillary itself, or more precisely the preparation of the capillary prior to analysis. Indeed, capillaries cut from the same roll and prepared exactly in the same way may exhibit dramatic differences in EOF. For example, some may be rapidly equilibrated while others may require very long equilibration time, such as 24 hours of many runs, before being stabilized. Furthermore, a capillary which is stabilized for a given buffer may not be directly usable with another buffer and thus has to be stabilized again. If the first buffer is used thereafter, the capillary may need to undergo yet another stabilization procedure. Schomburg writes in Guzman page 315, in a chapter dealing with the chemistry of surface modification, that: "It is of great analytical interest if, in practice, fused silica capillaries can be easily produced that have defined EOF properties and adsorption of analyte molecules. Capillaries of reproducible performance should be obtainable by a simple procedure of etching and rinsing. The process of equilibration before a capillary is conditioned for analytical separations should not require too much time. The aforementioned etching procedures are time-consuming and require long equilibration times before stable conditions for practical analyses are achieved. Therefore, it seems to be of interest to modify the silica surfaces by procedures, such as silanol derivation: ionic or non-ionic adsorption, as well as polymer coating. The time for equilibration between the buffer and the surfaces may be shortened, and the chemical properties of the coatings may be suited to suppress interaction or adsorption of special analyte molecules. The control of the electroosmotic flow and the suppression of analyte adsorption are the major aims of surface modifications."